Anti-neublastin antibodies and uses thereof

ABSTRACT

Antibodies and antigen binding fragments that bind to neublastin polypeptides are disclosed. Also disclosed are methods of using the antibodies and antigen binding fragments in assays for detecting the presence or amount of endogenous and/or exogenous neublastin in a sample and in methods of antagonizing neublastin bioactivity.

TECHNICAL FIELD

The invention relates to antibodies and antibody fragments that bind toneublastin.

BACKGROUND

Neublastin, also known as artemin and enovin, is a 24 kDa homodimeric,secreted protein that promotes the outgrowth and survival of neurons ofthe peripheral and central nervous system (Baudet et al., 2000,Development, 127:4335; Masure et al., 1999, Eur. J. Biochem., 266:892;Rosenblad et al., 2000, Mol. Cell Neurosci., 15(2):199). Neublastin mRNAis expressed predominantly in embryonic kidney and lung, and in adults,is expressed highest in pituitary gland, trachea, and placenta (Baudetet al., 2000, Development, 127:4335).

Neublastin is a member of the glial cell line-derived neurotrophicfactor (GDNF) ligand family. GDNF ligands activate both Ras andphosphatidylinositol-3-kinase signal transduction pathways by engagingthe membrane-bound c-RET receptor tyrosine kinase. This c-RET-mediatedsignaling requires an additional co-receptor, a glycosylphosphatidylinositol (GPI)-anchored GDNF family receptor alpha (GFRalpha) protein,which confers ligand specificity to c-RET. Four GFRalpha co-receptorproteins have been identified (GFRalpha-4). Neublastin shows highestaffinity for GFRalpha3 in vitro, however in studies using humanfibroblasts, neublastin can stimulate c-RET-dependent signaling througheither GFRalpha3 or GFRalpha1 (Baudet et al., 2000, Development,127:4335; Masure et al., 1999, Eur. J. Biochem. 266:892; Rosenblad etal., 2000, Mol. Cell Neurosci., 15(2):199).

The neublastinic-RET/GFRalpha3 ternary complex is localizedpredominantly to nociceptive sensory neurons that detect pain and injury(Orozco et al., 2001, Eur. J. Neurosci., 13(11):2177). Neublastin thushas potential clinical application in the treatment of neuropathy andmore specifically in the treatment of neuropathic pain. In addition,neublastin and GFRalpha3/RET are expressed at enhanced levels inpancreatic cancer tissues and neublastin promotes pancreatic cancer cellinvasion (Ceyhan et al., 2006, Annals of Surgery, 244:274).

SUMMARY

The invention is based, at least in part, on the discovery ofanti-neublastin antibodies that are useful in detecting endogenousand/or exogenous neublastin polypeptides and in antagonizing neublastinbioactivity.

In one aspect, the invention features an isolated antibody orantigen-binding fragment thereof that selectively binds to thepolypeptide of SEQ ID NO:1 on the same epitope as the antibody producedby the hybridoma deposited in the ATCC under Accession No. PTA-7624 orPTA-7625.

Also disclosed is an isolated antibody or antigen-binding fragmentthereof that selectively binds to the polypeptide of SEQ ID NO:1 andcrossblocks binding of the antibody produced by the hybridoma depositedin the ATCC under Accession No. PTA-7624 or PTA-7625.

Also disclosed is an antibody produced by the hybridoma deposited in theATCC under Accession No. PTA-7624 or PTA-7625. An antibody orantigen-binding fragment thereof can optionally contain theantigen-binding portion of an antibody produced by the hybridomadeposited in the ATCC under Accession No. PTA-7624 or PTA-7625.

The term “isolated” refers to a molecule that is substantially free ofits natural environment. For instance, an isolated antibody issubstantially free of cellular material from the cell or tissue sourcefrom which it was derived. The term also refers to preparations wherethe isolated antibody is sufficiently pure for a pharmaceuticalcomposition, or at least 70-80% (w/w) pure, at least 80-90% (w/w) pure,at least 90-95% (w/w) pure, or at least 95%, 96%, 97%, 98%, 99%, or 100%(w/w) pure.

The term “antibody or antigen-binding fragment thereof” encompassesproteins that include at least one immunoglobulin variable region, e.g.,an amino acid sequence that provides an immunoglobulin variable domainor immunoglobulin variable domain sequence. For example, the termincludes an antigen-binding protein that has a heavy (H) chain variableregion (abbreviated herein as VH), and a light (L) chain variable region(abbreviated herein as VL). In another example, the term includes anantigen binding protein that includes two heavy (H) chain variableregions and two light (L) chain variable regions. The term encompassesantigen-binding fragments of antibodies (e.g., single chain antibodies,Fab fragments, F(ab′)2 fragments, Fd fragments, Fv fragments, and dAbfragments) as well as complete antibodies, e.g., intact immunoglobulinsof types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof). Thelight chains of the immunoglobulin may be of types kappa or lambda. Insome embodiments, the antibody is glycosylated. An antibody can befunctional for antibody-dependent cytotoxicity and/orcomplement-mediated cytotoxicity, or may be non-functional for one orboth of these activities. The VH and VL regions can be furthersubdivided into regions of hypervariability, termed “complementaritydetermining regions” (“CDR”), interspersed with regions that are moreconserved, termed “framework regions” (FR). The extent of the FR's andCDR's has been precisely defined (see, Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, USDepartment of Health and Human Services, NIH Publication No. 91-3242;and Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917). Kabatdefinitions are used herein. Each VH and VL is typically composed ofthree CDR's and four FR's, arranged from amino-terminus tocarboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3,CDR3, FR4.

The term “selectively binds” refers to two molecules forming a complexthat is stable under physiologic conditions. Selective binding ischaracterized by a high affinity and a low to moderate capacity asdistinguished from nonspecific binding which usually has a low affinitywith a moderate to high capacity. Typically, binding is consideredselective when the antibody binds with a Kd of less than 10-6 M. Ifnecessary, nonspecific binding can be reduced without substantiallyaffecting selective binding by varying the binding conditions.

The term “crossblocking antibody” refers to a first anti-neublastinantibody that, when bound to a neublastin polypeptide, reduces oreliminates the ability of a second anti-neublastin antibody to bind tothe neublastin polypeptide (relative to binding of the secondanti-neublastin antibody to the neublastin polypeptide that occurs inthe absence of the first anti-neublastin antibody).

In some embodiments, an antibody or antigen-binding fragment thereofdescribed herein is a humanized antibody.

In some embodiments, an antibody or antigen-binding fragment thereofdescribed herein is a fully human antibody.

In some embodiments, an antibody or antigen-binding fragment thereofdescribed herein is a monoclonal antibody.

In some embodiments, an antibody or antigen-binding fragment thereofdescribed herein is a single chain antibody.

In some embodiments, an antibody or antigen-binding fragment thereofdescribed herein is a polyclonal antibody, a chimeric antibody, anF_(ab) fragment, an F_((ab′)2) fragment, an F_(ab′) fragment, an F_(sc)fragment, or an F_(v) fragment.

Also disclosed is an isolated cell that produces an antibody orantigen-binding fragment thereof described herein. The cell can be, forexample, a fused cell (e.g., a hybridoma) obtained by fusing a mammalianB cell and myeloma cell.

Also disclosed is a conjugate containing an antibody or antigen-bindingfragment thereof described herein linked to a detectable label. Thedetectable label can be, for example, horseradish peroxidase or alkalinephosphatase or a fluorescent or radio-labeled marker.

Also disclosed is a conjugate containing an antibody or antigen-bindingfragment thereof described herein linked to a solid-phase matrix (e.g.,a multi-well assay plate, sepharose, agarose, or a magnetic bead).

Also disclosed is a pharmaceutical composition containing (i) anantibody or antigen-binding fragment thereof described herein, and (ii)a pharmaceutically acceptable carrier.

In another aspect, the invention features a method of determining thepresence or amount of a neublastin polypeptide in a sample, which methodincludes: contacting a sample with an antibody or antigen-bindingfragment thereof described herein for a time sufficient for the antibodyor antigen-binding fragment thereof to bind to a neublastin polypeptide,if present in the sample; and detecting the presence or amount of theantibody or antigen-binding fragment thereof bound to the neublastinpolypeptide to thereby determine the presence or amount of theneublastin polypeptide in the sample. In some embodiments, the methodincludes additional steps of: prior to the detecting step, contactingthe sample with a second antibody or antigen-binding fragment thereofthat selectively binds to the polypeptide of SEQ ID NO:1 for a timesufficient for the second antibody or antigen-binding fragment thereofto bind to the neublastin polypeptide bound to an antibody orantigen-binding fragment thereof described herein; and detecting thepresence or amount of the second antibody or antigen-binding fragmentthereof bound to the neublastin polypeptide.

Also disclosed is a method of determining the presence or amount of aneublastin polypeptide in a sample, which method includes: contacting asample with a first antibody or antigen-binding fragment thereof thatselectively binds to the polypeptide of SEQ ID NO:1 on the same epitopeas the antibody produced by the hybridoma deposited in the ATCC underAccession No. PTA-7625 for a time sufficient for the first antibody orantigen-binding fragment thereof to bind to a neublastin polypeptide, ifpresent in the sample; contacting the sample with a second antibody orantigen-binding fragment thereof that selectively binds to thepolypeptide of SEQ ID NO:1 on the same epitope as the antibody producedby the hybridoma deposited in the ATCC under Accession No. PTA-7624 fora time sufficient for the second antibody or antigen-binding fragmentthereof to bind the neublastin polypeptide, if bound to the firstantibody or antigen-binding fragment thereof; and detecting the presenceor amount of the second antibody or antigen-binding fragment thereofbound to the neublastin polypeptide, to thereby determine the presenceor amount of the neublastin polypeptide in the sample.

In some embodiments, the first antibody or antigen-binding fragmentthereof selectively binds to the polypeptide of SEQ ID NO:1 andcrossblocks binding of the antibody produced by the hybridoma depositedin the ATCC under Accession No. PTA-7625. In some embodiments, the firstantibody is produced by the hybridoma deposited in the ATCC underAccession No. PTA-7625.

In some embodiments, the second antibody or antigen-binding fragmentthereof selectively binds to the polypeptide of SEQ ID NO:1 andcrossblocks binding of the antibody produced by the hybridoma depositedin the ATCC under Accession No. PTA-7624. In some embodiments, thesecond antibody is produced by the hybridoma deposited in the ATCC underAccession No. PTA-7624.

Also disclosed is a method of determining the presence or amount of aneublastin polypeptide in a sample, which method includes: contacting asample with a first antibody or antigen-binding fragment thereof thatselectively binds to the polypeptide of SEQ ID NO:1 on the same epitopeas the antibody produced by the hybridoma deposited in the ATCC underAccession No. PTA-7625 for a time sufficient for the first antibody orantigen-binding fragment thereof to bind to a neublastin polypeptide, ifpresent in the sample; contacting the sample with a second antibody orantigen-binding fragment thereof that selectively binds to thepolypeptide of SEQ ID NO:1 on the same epitope as the antibody producedby the hybridoma deposited in the ATCC under Accession No. PTA-7625 fora time sufficient for the second antibody or antigen-binding fragmentthereof to bind to the neublastin polypeptide, if bound to the firstantibody or antigen-binding fragment thereof; and detecting the presenceor amount of the second antibody or antigen-binding fragment thereofbound to the neublastin polypeptide, to thereby determine the presenceor amount of the neublastin polypeptide in the sample.

In some embodiments, the first antibody or antigen-binding fragmentthereof selectively binds to the polypeptide of SEQ ID NO:1 andcrossblocks binding of the antibody produced by the hybridoma depositedin the ATCC under Accession No. PTA-7625. In some embodiments, the firstantibody is produced by the hybridoma deposited in the ATCC underAccession No. PTA-7625.

In some embodiments, the second antibody or antigen-binding fragmentthereof selectively binds to the polypeptide of SEQ ID NO:1 andcrossblocks binding of the antibody produced by the hybridoma depositedin the ATCC under Accession No. PTA-7625. In some embodiments, thesecond antibody is produced by the hybridoma deposited in the ATCC underAccession No. PTA-7625.

The methods described herein for determining the presence or amount of aneublastin polypeptide in a sample can optionally be performed as asandwich-based immunoassay.

In any of the methods described herein, the sample can be obtained froma mammal (e.g., a human). The neublastin polypeptide detected accordingto the methods can be, for example, endogenous, wild-type neublastin oran exogenous, recombinant neublastin.

The neublastin polypeptide detected in any of the methods describedherein can contain (or consist of) an amino acid sequence that is atleast 80% identical (at least 90%, 95%, or 98% identical) to amino acids15-113 of SEQ ID NO:1, wherein the polypeptide, when dimerized, binds toa complex containing GFRalpha3 and RET. In some embodiments, theneublastin polypeptide contains (or consists of) amino acids 15-113 ofSEQ ID NO:1, amino acids 15-113 of SEQ ID NO:2, amino acids 15-113 ofSEQ ID NO:3, amino acids 15-113 of SEQ ID NO:4, amino acids 15-113 ofSEQ ID NO:5, amino acids 15-113 of SEQ ID NO:8, or amino acids 15-113 ofSEQ ID NO:9. In some embodiments, the neublastin polypeptide contains(or consists of) the amino acid sequence of SEQ ID NO:1, the amino acidsequence of SEQ ID NO:2, the amino acid sequence of SEQ ID NO:3, theamino acid sequence of SEQ ID NO:4, the amino acid sequence of SEQ IDNO:5, the amino acid sequence of SEQ ID NO:8, or the amino acid sequenceof SEQ ID NO:9. In some embodiments, the neublastin polypeptide contains(or consists of) amino acids 10-113 of SEQ ID NO:1.

The sample used in any of the methods described herein can be obtainedfrom a mammal (e.g., a human) to which the neublastin polypeptide (e.g.,a recombinant neublastin polypeptide) has previously been administered(e.g., by subcutaneous or intravenous administration). For example, thesample can be obtained from blood, serum, saliva, semen, urine, lacrimalfluid, or cerebral spinal fluid.

Also disclosed is a method of antagonizing neublastin activity byadministering to a mammal an amount of an antibody or antigen-bindingfragment thereof described herein effective to reduce or eliminate theability of endogenous, wild-type neublastin to bind to a complexcontaining RET and GFRalpha3 and induce dimerization andautophosphorylation of RET. Also disclosed is the use of an antibody orantigen-binding fragment thereof described herein for the preparation ofa pharmaceutical composition for antagonizing neublastin activity (byreducing or eliminating the ability of endogenous, wild-type neublastinto bind to a complex containing RET and GFRalpha3 and inducedimerization and autophosphorylation of RET).

Also disclosed is a method of treating a cancer by administering to amammal having a cancer (e.g., a pancreatic cancer) a pharmaceuticalcomposition containing a therapeutically effective amount of an antibodyor antigen-binding fragment thereof described herein. Also disclosed isthe use of an antibody or antigen-binding fragment thereof describedherein for the preparation of a pharmaceutical composition for treatinga cancer (e.g., a pancreatic cancer).

As used herein, the terms “to treat,” “treating,” and “treatment” referto administering a therapy in an amount, manner, and/or mode effectiveto improve or ameliorate a symptom or parameter that characterizes apathological condition, to reduce the severity of a symptom or parameterthat characterizes a pathological condition, to prevent, slow or reverseprogression of the pathological condition, or to prevent one or moresymptom or parameter of the pathological condition.

The mammal treated according to the methods and uses described hereincan be, e.g., a human, a mouse, a rat, a cow, a pig, a dog, a cat, or amonkey.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the present invention, the exemplary methods andmaterials are described below. All publications, patent applications,patents, and other references mentioned herein are incorporated byreference in their entirety. In case of conflict, the presentapplication, including definitions, will control. The materials,methods, and examples are illustrative only and not intended to belimiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is an alignment of wild-type human (SEQ ID NO:10), mouse (SEQ IDNO:11), and rat (SEQ ID NO:12) pre pro neublastin polypeptides. The leftand right vertical lines indicate, respectively, the start of the mature113 amino and 104 amino acid forms. The RRXR heparin binding motif isboxed.

FIG. 2 is a graph depicting the detection of standard concentrations ofneublastin using the P3B3 assay.

FIG. 3 is a graph depicting the linear portion of the P3B3 assay againstvarious standard concentrations of neublastin.

FIG. 4 is a graph depicting the kinetics of exogenous human neublastin(hNBN104 expressed in CHO cells) in rat serum following subcutaneousadministration.

FIG. 5 is a scatter plot depicting the ability of anti-hNBN monoclonalantibodies to inhibit NBN-mediated c-RET phosphorylation in murinecells.

DETAILED DESCRIPTION

Disclosed are antibodies and antigen-binding fragments thereof that bindto neublastin. The anti-neublastin antibodies are useful in assays todetect endogenous and/or exogenous neublastin polypeptides. In addition,the antibodies can be used to antagonize neublastin bioactivity andtreat cancers such as pancreatic cancer.

Antibody Generation

Antibodies or antibody fragments that bind to neublastin can begenerated by immunization, e.g., using an animal, or by in vitro methodssuch as phage display. A polypeptide that includes all or part ofneublastin can be used to generate an antibody or antibody fragment.Mature, full length wild-type human neublastin contains the followingamino acid sequence: AGGPGSRARAAGARGCRLRSQLVPVRALGLGHRSDELVRFRFCSGSCRRARSPHDLSLASLLGAGALRPPPGSRPVSQPCCRPTRYEAVSFMDVNSTWRTVDRLSATACGCLG (SEQ ID NO:1). The full length mature neublastinpolypeptide of SEQ ID NO:1 or a portion thereof (e.g., an amino terminaltruncation containing the 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, 100, 105, 110, 111, or 112 carboxy terminal amino acidsof the polypeptide of SEQ ID NO:1) can be used as an immunogen togenerate antibodies that can be screened for reactivity to neublastin.Alternatively, a cell expressing all or part of neublastin can be usedas an immunogen to generate antibodies.

In some embodiments, an immunized animal contains immunoglobulinproducing cells with natural, human, or partially human immunoglobulinloci. In some embodiments, the non-human animal includes at least a partof a human immunoglobulin gene. For example, it is possible to engineermouse strains that are deficient in mouse antibody production andcontain large fragments of the human Ig loci. Using hybridomatechnology, antigen-specific monoclonal antibodies derived from thegenes with the desired specificity can be produced and selected. See,e.g., XenoMouse™, Green et al. Nature Genetics 7:13-21 (1994), US2003-0070185, U.S. Pat. No. 5,789,650, and WO 96/34096.

Non-human antibodies to neublastin can also be produced, e.g., in arodent. The non-human antibody can be humanized, e.g., as described inU.S. Pat. No. 6,602,503, EP 239 400, U.S. Pat. No. 5,693,761, and U.S.Pat. No. 6,407,213.

EP 239 400 (Winter et al.) describes altering antibodies by substitution(within a given variable region) of their CDRs for one species withthose from another. CDR-substituted antibodies can be less likely toelicit an immune response in humans compared to true chimeric antibodiesbecause the CDR-substituted antibodies contain considerably lessnon-human components. See Riechmann et al., 1988, Nature 332, 323-327;Verhoeyen et al., 1988, Science 239, 1534-1536. Typically, CDRs of amurine antibody are substituted into the corresponding regions in ahuman antibody by using recombinant nucleic acid technology to producesequences encoding the desired substituted antibody. Human constantregion gene segments of the desired isotype (e.g., gamma I for CH andkappa for CL) can be added and the humanized heavy and light chain genescan be co-expressed in mammalian cells to produce soluble humanizedantibody.

WO 90/07861 describes a process that includes choosing human V frameworkregions by computer analysis for optimal protein sequence homology tothe V region framework of the original murine antibody, and modeling thetertiary structure of the murine V region to visualize framework aminoacid residues that are likely to interact with the murine CDRs. Thesemurine amino acid residues are then superimposed on the homologous humanframework. See also U.S. Pat. Nos. 5,693,762; 5,693,761; 5,585,089; and5,530,101. Tempest et al., 1991, Biotechnology 9, 266-271 use, asstandard, the V region frameworks derived from NEWM and REI heavy andlight chains, respectively, for CDR-grafting without radicalintroduction of mouse residues. An advantage of using the Tempest et al.approach to construct NEWM and REI based humanized antibodies is thatthe three dimensional structures of NEWM and REI variable regions areknown from x-ray crystallography and thus specific interactions betweenCDRs and V region framework residues can be modeled.

Non-human antibodies can be modified to include substitutions thatinsert human immunoglobulin sequences, e.g., consensus human amino acidresidues at particular positions, e.g., at one or more (e.g., at leastfive, ten, twelve, or all) of the following positions: (in the frameworkof the variable domain of the light chain) 4L, 35L, 36L, 38L, 43L, 44L,58L, 46L, 62L, 63L, 64L, 65L, 66L, 67L, 68L, 69L, 70L, 71L, 73L, 85L,87L, 98L, and/or (in the framework of the variable domain of the heavychain) 2H, 4H, 24H, 36H, 37H, 39H, 43H, 45H, 49H, 58H, 60H, 67H, 68H,69H, 70H, 73H, 74H, 75H, 78H, 91H, 92H, 93H, and/or 103H (according tothe Kabat numbering). See, e.g., U.S. Pat. No. 6,407,213.

Fully human monoclonal antibodies that bind to neublastin can beproduced, e.g., using in vitro-primed human splenocytes, as described byBoerner et al., 1991, J. Immunol., 147, 86-95. They may be prepared byrepertoire cloning as described by Persson et al., 1991, Proc. Nat.Acad. Sci. USA, 88: 2432-2436 or by Huang and Stollar, 1991, J. Immunol.Methods 141, 227-236; also U.S. Pat. No. 5,798,230. Large nonimmunizedhuman phage display libraries may also be used to isolate high affinityantibodies that can be developed as human therapeutics using standardphage technology (see, e.g., Vaughan et al, 1996; Hoogenboom et al.(1998) Immunotechnology 4:1-20; and Hoogenboom et al. (2000) ImmunolToday 2:371-8; US 2003-0232333).

As used herein, an “immunoglobulin variable domain sequence” refers toan amino acid sequence that can form the structure of an immunoglobulinvariable domain. For example, the sequence may include all or part ofthe amino acid sequence of a naturally-occurring variable domain. Forexample, the sequence may omit one, two or more N- or C-terminal aminoacids, internal amino acids, may include one or more insertions oradditional terminal amino acids, or may include other alterations. Inone embodiment, a polypeptide that includes an immunoglobulin variabledomain sequence can associate with another immunoglobulin variabledomain sequence to form a target binding structure (or “antigen bindingsite”), e.g., a structure that interacts with Neublastin.

The VH or VL chain of the antibody can further include all or part of aheavy or light chain constant region, to thereby form a heavy or lightimmunoglobulin chain, respectively. In one embodiment, the antibody is atetramer of two heavy immunoglobulin chains and two light immunoglobulinchains. The heavy and light immunoglobulin chains can be connected bydisulfide bonds. The heavy chain constant region typically includesthree constant domains, CH1, CH2 and CH3. The light chain constantregion typically includes a CL domain. The variable region of the heavyand light chains contains a binding domain that interacts with anantigen. The constant regions of the antibodies typically mediate thebinding of the antibody to host tissues or factors, including variouscells of the immune system (e.g., effector cells) and the firstcomponent (Clq) of the classical complement system.

One or more regions of an antibody can be human, effectively human, orhumanized. For example, one or more of the variable regions can be humanor effectively human. For example, one or more of the CDRs, e.g., heavychain (HC) CDR1, HC CDR2, HC CDR3, light chain (LC) CDR1, LC CDR2, andLC CDR3, can be human. Each of the light chain CDRs can be human. HCCDR3 can be human. One or more of the framework regions (FR) can behuman, e.g., FR1, FR2, FR3, and FR4 of the HC or LC. In someembodiments, all the framework regions are human, e.g., derived from ahuman somatic cell, e.g., a hematopoietic cell that producesimmunoglobulins or a non-hematopoietic cell. In one embodiment, thehuman sequences are germline sequences, e.g., encoded by a germlinenucleic acid. One or more of the constant regions can be human,effectively human, or humanized. In another embodiment, at least 70, 75,80, 85, 90, 92, 95, or 98% of the framework regions (e.g., FR1, FR2, andFR3, collectively, or FR1, FR2, FR3, and FR4, collectively) or theentire antibody can be human, effectively human, or humanized. Forexample, FR1, FR2, and FR3 collectively can be at least 70, 75, 80, 85,90, 92, 95, 98, or 99% identical to a human sequence encoded by a humangermline segment.

An “effectively human” immunoglobulin variable region is animmunoglobulin variable region that includes a sufficient number ofhuman framework amino acid positions such that the immunoglobulinvariable region does not elicit an immunogenic response in a normalhuman. An “effectively human” antibody is an antibody that includes asufficient number of human amino acid positions such that the antibodydoes not elicit an immunogenic response in a normal human.

A “humanized” immunoglobulin variable region is an immunoglobulinvariable region that is modified such that the modified form elicitsless of an immune response in a human than does the non-modified form,e.g., is modified to include a sufficient number of human frameworkamino acid positions such that the immunoglobulin variable region doesnot elicit an immunogenic response in a normal human. Descriptions of“humanized” immunoglobulins include, for example, U.S. Pat. No.6,407,213 and U.S. Pat. No. 5,693,762. In some cases, humanizedimmunoglobulins can include a non-human amino acid at one or moreframework amino acid positions.

All or part of an antibody can be encoded by an immunoglobulin gene or asegment thereof. Exemplary human immunoglobulin genes include the kappa,lambda, alpha (IgA1 and IgA2), gamma (IgG1, IgG2, IgG3, IgG4), delta,epsilon and mu constant region genes, as well as the myriadimmunoglobulin variable region genes. Full-length immunoglobulin “lightchains” (about 25 Kd or 214 amino acids) are encoded by a variableregion gene at the NH2-terminus (about 110 amino acids) and a kappa orlambda constant region gene at the COOH-terminus. Full-lengthimmunoglobulin “heavy chains” (about 50 Kd or 446 amino acids), aresimilarly encoded by a variable region gene (about 116 amino acids) andone of the other aforementioned constant region genes, e.g., gamma(encoding about 330 amino acids).

The term “antigen-binding fragment” of a full length antibody refers toone or more fragments of a full-length antibody that retain the abilityto specifically bind to a target of interest (i.e., neublastin).Examples of binding fragments encompassed within the term“antigen-binding fragment” of a full length antibody include: (i) a Fabfragment, a monovalent fragment consisting of the VL, VH, CL and CH1domains; (ii) a F(ab′)2 fragment, a bivalent fragment including two Fabfragments linked by a disulfide bridge at the hinge region; (iii) a Fdfragment consisting of the VH and CH1 domains; (iv) a Fv fragmentconsisting of the VL and VH domains of a single arm of an antibody; (v)a dAb fragment (Ward et al., (1989) Nature 341:544-546), which consistsof a VH domain; and (vi) an isolated complementarity determining region(CDR) that retains functionality. Furthermore, although the two domainsof the Fv fragment, VL and VH, are coded for by separate genes, they canbe joined, using recombinant methods, by a synthetic linker that enablesthem to be made as a single protein chain in which the VL and VH regionspair to form monovalent molecules known as single chain Fv (scFv). Seee.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883.

Deposits

Hybridomas producing the monoclonal antibody P1H1.G7 and the monoclonalantibody P3B3.6 have been deposited with the American Type CultureCollection (ATCC) under the terms of the Budapest Treaty on theInternational Recognition of the Deposit of Microorganisms for thePurpose of Patent Procedure on Jun. 1, 2006 and bear the accessionnumbers ATCC PTA-7624 (P1H1.G7) and ATCC PTA-7625 (P3B3.6). Applicantsacknowledge their duty to replace the deposits should the depository beunable to furnish a sample when requested due to the condition of thedeposit before the end of the term of a patent issued hereon. Applicantsalso acknowledge their responsibility to notify the ATCC of the issuanceof such a patent, at which time the deposit will be made available tothe public. Prior to that time, the deposit will be made available tothe Commissioner of Patents under the terms of 37 C.F.R. §1.14 and 35U.S.C. §112.

Neublastin Polypeptides

Mature wild-type human neublastin is 113 amino acids in length andconsists of the amino acid sequence depicted in SEQ ID NO:1. Theantibodies described herein are useful in detecting endogenous and/orexogenous neublastin polypeptides. Endogenous neublastin can be, forexample, the mature, wild-type human neublastin of SEQ ID NO:1,unprocessed neublastin, or a neublastin polypeptide at one of severalstages of processing. In addition, or alternatively, the anti-neublastinantibodies described herein can be used to detect an exogenousneublastin polypeptide. Exogenous neublastin can contain the amino acidsequence of SEQ ID NO:1 (i.e., a sequence identical to that of naturallyoccurring wild-type human neublastin) or a biologically active variantthereof. A variant neublastin polypeptide can contain one or moreadditions, substitutions, and/or deletions, as detailed in the followingsections. Wild-type neublastin polypeptides and biologically activevariants thereof are collectively referred to herein as “neublastinpolypeptides.”

A variant neublastin polypeptide can vary in length from thecorresponding wild-type polypeptide. Although the mature humanneublastin polypeptide (SEQ ID NO:1) consists of the carboxy terminal113 amino acids of pre pro neublastin (SEQ ID NO:10), not all of the 113amino acids are required to achieve useful neublastin biologicalactivity. Amino terminal truncation is permissible. Thus, a variantneublastin polypeptide can contain, for example, the carboxy terminal99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or113 amino acids of SEQ ID NO:1 (i.e., its length can be 99, 100, 101,102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, or 113 aminoacids).

A variant neublastin polypeptide can also vary in sequence from thecorresponding wild-type polypeptide. In particular, certain amino acidsubstitutions can be introduced into the neublastin sequence withoutappreciable loss of a neublastin biological activity. In exemplaryembodiments, a variant neublastin polypeptide (i) contains one or moreamino acid substitutions, and (ii) is at least 70%, 80%, 85%, 90%, 95%,98% or 99% identical to SEQ ID NO:1 (or 70%, 80%, 85%, 90%, 95%, 98% or99% identical to amino acids 15-113 of SEQ ID NO:1). A variantneublastin polypeptide differing in sequence from SEQ ID NO:1 (ordiffering in sequence from amino acids 15-113 of SEQ ID NO:1) caninclude one or more amino acid substitutions (conservative ornon-conservative), one or more deletions, and/or one or more insertions.

FIG. 1 is an alignment of the wild-type human, mouse, and rat pre proneublastin polypeptides. The vertical lines in FIG. 1 indicate the startof the mature 113 amino acid form (left vertical line) and 104 aminoacid form (right vertical line) of neublastin. The RRXR heparin bindingmotif is boxed. This alignment of naturally occurring, bioactive formsof neublastin indicates specific exemplary residues (i.e., those thatare not conserved among the human, mouse, and rat forms) that can besubstituted without eliminating bioactivity.

Percent identity between amino acid sequences can be determined usingthe BLAST 2.0 program. Sequence comparison can be performed using anungapped alignment and using the default parameters (Blossom 62 matrix,gap existence cost of 11, per residue gap cost of 1, and a lambda ratioof 0.85). The mathematical algorithm used in BLAST programs is describedin Altschul et al., 1997, Nucleic Acids Research 25:3389-3402.

A conservative substitution is the substitution of one amino acid foranother with similar characteristics. Conservative substitutions includesubstitutions within the following groups: valine, alanine and glycine;leucine, valine, and isoleucine; aspartic acid and glutamic acid;asparagine and glutamine; serine, cysteine, and threonine; lysine andarginine; and phenylalanine and tyrosine. The non-polar hydrophobicamino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid. Any substitution of one memberof the above-mentioned polar, basic or acidic groups by another memberof the same group can be deemed a conservative substitution.

Non-conservative substitutions include those in which (i) a residuehaving an electropositive side chain (e.g., Arg, His or Lys) issubstituted for, or by, an electronegative residue (e.g., Glu or Asp),(ii) a hydrophilic residue (e.g., Ser or Thr) is substituted for, or by,a hydrophobic residue (e.g., Ala, Leu, Ile, Phe or Val), (iii) acysteine or proline is substituted for, or by, any other residue, or(iv) a residue having a bulky hydrophobic or aromatic side chain (e.g.,Val, Ile, Phe or Trp) is substituted for, or by, one having a smallerside chain (e.g., Ala, Ser) or no side chain (e.g., Gly).

A biologically active variant neublastin polypeptide, when dimerized,binds to a ternary complex containing GFRalpha3 and RET. Any method fordetecting binding to this complex can be used to evaluate the biologicalactivity a variant neublastin polypeptide. Exemplary assays fordetecting the ternary complex-binding ability of a variant neublastinpolypeptide are described in WO00/01815 (the content of which isincorporated herein by reference).

A variant neublastin polypeptide can also be assessed to evaluate itsability to trigger the neublastin signaling cascade. For example, theKinase Receptor Activation (KIRA) assay can be used to assess theability of a variant neublastin polypeptide to induce RETautophosphorylation (See also, Sadick et al., 1996, Anal. Biochem.,235(2):207).

Substitutions at one or more of the following amino acid residues areexpected to result in a variant neublastin polypeptide having reduced orabsent heparin binding ability as compared to wild-type neublastin: Arg48, Arg 49, Arg 51, Ser 46, Ser 73, Gly 72, Arg 39, Gln 21, Ser 20, Arg68, Arg 33, His 32, Val 94, Arg 7, Arg 9, or Arg 14. Reference to aneublastin amino acid reside by position number refers to the numberingof residues relative to SEQ ID NO:1. A neublastin amino acid residuedesignated for substitution (e.g., an arginine residue at position 48,49, and/or 51) can be substituted with a non-conservative amino acidresidue (e.g., glutamic acid) or a conservative or amino acid residue.Exemplary amino acids that can be substituted at a residue identifiedherein (e.g., position 48, 49, and/or 51) include glutamic acid,aspartic acid, and alanine.

Examples of variant neublastin polypeptides that exhibit reduced orabsent heparin binding are disclosed in Table 1 and in WO 2006/023781(the content of which is incorporated herein by reference). Amino acidresidues of the variant neublastin polypeptides that are mutated ascompared to the corresponding wild-type position are bolded andunderlined in Table 1. In addition, the neublastin polypeptide (e.g.,113, 99, or 104 amino acids in length) used as the background for thesubstitution is depicted in Table 1.

TABLE 1 Variant Neublastin Polypeptides SEQ Position Length ofAmino Acid  ID NO Substituted Polypeptide Sequence 2 Arg 48 113AGGPGSRARAAGARGCRL RSQLVPVRALGLGHRSDE LVRFRFCSGSC E RARSPHDLSLASLLGAGALRPPPG SRPVSQPCCRPTRYEAVS FMDVNSTWRTVDRLSATA CGCLG 3 Arg 49113 AGGPGSRARAAGARGCRL RSQLVPVRALGLGHRSDE LVRFRFCSGSCREARSPHDLSLASLLGAGALRPPPG SRPVSQPCCRPTRYEAVS FMDVNSTWRTVDRLSATA CGCLG 4 Arg 51113 AGGPGSRARAAGARGCRL RSQLVPVRALGLGHRSDE LVRFRFCSGSCRRAESPHDLSLASLLGAGALRPPPG SRPVSQPCCRPTRYEAVS FMDVNSTWRTVDRLSATA CGCLG 5Arg 48 and 113 AGGPGSRARAAGARGCRL Arg 49 RSQLVPVRALGLGHRSDE LVRFRFCSGSCEE ARSPH DLSLASLLGAGALRPPPG SRPVSQPCCRPTRYEAVS FMDVNSTWRTVDRLSATA CGCLG6 Arg 48 and 99 GCRLRSQLVPVRALGLGH Arg 49 RSDELVRFRFCSGSC EE ARSPHDLSLASLLGAGALR PPPGSRPVSQPCCRPTRY EAVSFMDVNSTWRTVDRL SATACGCLG 7Arg 48 and 104 AAGARGCRLRSQLVPVRA Arg 49 LGLGHRSDELVRFRFCSG SC EEARSPHDLSLASLLG AGALRPPPGSRPVSQPCC RPTRYEAVSFMDVNSTWR TVDRLSATACGCLG 8Arg 49 and 113 AGGPGSRARAAGARGCRL Arg 51 RSQLVPVRALGLGHRSDE LVRFRFCSGSCRE A E SPH DLSLASLLGAGALRPPPG SRPVSQPCCRPTRYEAVS FMDVNSTWRTVDRLSATA CGCLG9 Arg 48 and 113 AGGPGSRARAAGARGCRL Arg 51 RSQLVPVRALGLGHRSDELVRFRFCSGSC E RA E SPH DLSLASLLGAGALRPPPG SRPVSQPCCRPTRYEAVSFMDVNSTWRTVDRLSATA CGCLG

A neublastin polypeptide can be optionally coupled to a polymer (e.g., apolyalkylene glycol moiety such as a polyethylene glycol moiety). Insome embodiments, the polymer is coupled to the polypeptide at a site onthe neublastin polypeptide that is an N terminus. In some embodiments, avariant neublastin polypeptide includes at least one amino acidsubstitution with respect to SEQ ID NO:1 (or with respect to amino acids15-113 of SEQ ID NO:1), which provides an internal polymer conjugationsite to which a polymer can be conjugated. In some embodiments, thepolymer is coupled to a variant neublastin polypeptide at a residue(numbered in accordance with the sequence of SEQ ID NO:1) selected fromthe group consisting of position 14, position 39, position 68, andposition 95. Exemplary neublastin variants that provide internal polymerconjugation sites are described in WO 02/060929 and WO 04/069176 (thecontents of which are incorporated herein by reference).

A polypeptide can optionally contain heterologous amino acid sequencesin addition to a neublastin polypeptide. “Heterologous,” as used whenreferring to an amino acid sequence, refers to a sequence thatoriginates from a source foreign to the particular host cell, or, iffrom the same host cell, is modified from its original form. Exemplaryheterologous sequences include a heterologous signal sequence (e.g.,native rat albumin signal sequence, a modified rat signal sequence, or ahuman growth hormone signal sequence) or a sequence used forpurification of a neublastin polypeptide (e.g., a histidine tag).Examples of polypeptides containing a neublastin polypeptide and aheterologous signal sequence are described in US 20050158824 and WO2004/108760.

A naturally occurring or recombinantly produced neublastin polypeptidecan be isolated from cells or tissue sources using standard proteinpurification techniques. Alternatively, a mutated neublastin polypeptidecan be synthesized chemically using standard peptide synthesistechniques. The synthesis of short amino acid sequences is described in,e.g., Stewart, et al., Solid Phase Peptide Synthesis (2d ed., 1984).

A neublastin polypeptide can be produced by recombinant DNA techniques.For example, a nucleic acid molecule encoding a neublastin polypeptidecan be inserted into a vector, e.g., an expression vector, and thenucleic acid can be introduced into a cell. Suitable cells include,e.g., mammalian cells (such as human cells or CHO cells), fungal cells,yeast cells, insect cells, and bacterial cells (e.g., E. coli). Whenexpressed in a recombinant cell, the cell can be cultured underconditions allowing for expression of a neublastin polypeptide. Theneublastin polypeptide can be recovered from a cell suspension ifdesired. As used herein, “recovered” means that the polypeptide isremoved from those components of a cell or culture medium in which it ispresent prior to the recovery process. The recovery process can includeone or more refolding or purification steps. Buffers and methods forinducing folding of a denatured neublastin polypeptide are described in,e.g., WO 2006/023782.

Variant neublastin polypeptides can be constructed using any of severalmethods known in the art. One such method is site-directed mutagenesis,in which a specific nucleotide (or, if desired a small number ofspecific nucleotides) is changed in order to change a single amino acid(or, if desired, a small number of predetermined amino acid residues) inthe encoded variant neublastin polypeptide. Many site-directedmutagenesis kits are commercially available. One such kit is the“Transformer Site Directed Mutagenesis Kit” sold by ClontechLaboratories (Palo Alto, Calif.).

Neublastin Detection

The anti-neublastin antibodies described herein can be used inimmunoassay methods to detect a neublastin polypeptide in a sample.Depending upon the anti-neublastin antibody or combination ofanti-neublastin antibodies used, an assay can detect endogenousneublastin and/or an exogenous neublastin (e.g., a recombinantneublastin polypeptide). General immunoassay techniques are describedin, for example, Voller et al. (1978) J. Clin. Path. 31:507-20, andCrowther (1995) Methods in Mol. Biol., Vol 42.

The anti-neublastin antibodies described herein can be used insolid-phase immunoassays. Solid-phase immunoassays generally involve theadherence or conjugation of one component (e.g., the antibody or theantigen) of the immunoassay to a solid-phase matrix (e.g., sepharose,agarose, magnetic beads, or a multi-well assay plate). For example, aneublastin polypeptide in a sample can be adhered to a solid phasematrix. Non-adherent proteins are removed by subsequent washing steps.Adherent neublastin polypeptide can then be detected by the addition ofa detection antibody specific for a neublastin polypeptide. Thisdetection antibody can be, for example, a P1H1.G7 or P3B3.6anti-neublastin antibody described herein. In some embodiments, theantibodies can be directly coupled to a “detection moiety.” Detectionmoieties include, for example, fluorescent labels (e.g., cy5, cy3, greenfluorescent protein, or fluorescein). Detection moieties can also beradioisotope labels, such as ³⁵S, ³²P, or ¹²⁵I. Detection labels canalso be enzymes, e.g., alkaline phosphatase (AP), horseradish peroxidase(HRP), luciferase, or chloramphenicol acetyl transferase (CAT).Alternatively, it is often useful that the detection moiety be coupledto a secondary antibody that specifically recognizes the first,detection antibody (for example, to amplify the assay signal strength).In another embodiment, the detection antibody or secondary antibody canbe conjugated to a first member of a binding pair (e.g., biotin orstreptavidin) and the detection moiety can be linked to a second memberof a binding pair (e.g., streptavidin or biotin).

A solid-phase immunoassay can be a “sandwich” type immunoassay, whereina first anti-neublastin antibody (e.g., P1H1.G7 or P3B3.6) is adhered toa solid-phase matrix (e.g., sepharose, agarose, magnetic beads, or amulti-well assay plate). A neublastin polypeptide-containing sample isthen added to the antibody-coupled matrix and incubated for a timesufficient to allow binding of the neublastin polypeptide (if present)to the immobilized anti-neublastin antibody. Unbound polypeptides areremoved in subsequent wash steps. Neublastin polypeptides, if bound bythe immobilized anti-neublastin antibody, can then be detected using asecond anti-neublastin antibody. The second antibody can optionally havea different epitope specificity than the first antibody (e.g., the firstantibody binds to the same epitope as P1H1.G7 and the second antibodybinds to the same epitope as P3B3.6). Alternatively, the twoanti-neublastin antibodies can have overlapping or identical epitopespecificity (e.g., both antibodies bind to the same epitope as P3B3.6).As indicated above, the second anti-neublastin antibody can be directlycoupled to a detection moiety or, alternatively, the detection moietycan be conjugated to a “secondary” antibody that is capable ofrecognizing the second detection antibody of the immunoassay method.

A sandwich-type immunoassay can be used to detect the presence ofexogenous neublastin in a sample (e.g., a sample taken from a subjectthat had previously been administered a recombinant neublastinpolypeptide). In an example of such an assay, the first anti-neublastin“capture” antibody is the P3B3.6 antibody or an antibody that binds tothe same epitope as P3B3.6 and the second anti-neublastin “detection”antibody is the P3B3.6 antibody or an antibody that binds to the sameepitope as P3B3.6. An alternative sandwich-type immunoassay can be usedto detect both endogenous and exogenous neublastin in a sample. In anexample of such an assay, the first anti-neublastin “capture” antibodyis the P3B3.6 antibody or an antibody that binds to the same epitope asP3B3.6 and the second anti-neublastin “detection” antibody is theP1H1.G7 antibody or an antibody that binds to the same epitope asP1H1.G7.

A neublastin polypeptide can be detected via a competition-basedimmunoassay. Competitive inhibition assay formats generally entail thesimultaneous addition of labeled analyte (e.g., detectable-labelconjugated neublastin) and unlabeled analyte (e.g., neublastin from thesample). Both labeled and unlabeled analyte then compete simultaneouslyfor the binding site on the capture antibody on the plate. Like thesequential competitive inhibition format, the colored signal isinversely proportional to the concentration of unlabeled target analytein the sample. As above, anti-neublastin antibody is adhered to a solidphase matrix. The neublastin-containing sample, before addition to theantibody, is premixed with a known (i.e., standard) amount of neublastincompetitor conjugated to a detection moiety (e.g., a “detection moiety”as described herein). Both the sample neublastin polypeptide and theneublastin-detection standard are incubated with the antibody for a timesufficient for the antibody to bind to neublastin in the sample and/orthe standard. Binding of the neublastin-detection moiety standard to theantibody, and the detection signal produced by the assay, is dependenton the level of neublastin present in the sample (i.e., the higher theamount of neublastin polypeptide in a sample, the lower the signalproduced in the assay). Quantitation of the amount of neublastin in asample, can be determined by comparing the amount of signal produced bythe neublastin-detection moiety standard in the absence of sampleneublastin polypeptide relative to the signal produced from samples inwhich sample neublastin is mixed with the standard. An example of acompetition-based immunoassay is the ORIGEN assay (Igen, Inc.,Rockville, Md.).

A neublastin polypeptide can be detected by western blotting using theanti-neublastin antibodies described herein. Western blotting methodsare described in, for example, Sambrook et al. (2001) Molecular Cloning,a Lab Manual, 3^(rd) Edition. A sample containing a neublastinpolypeptide can be suspended in a denaturing buffer (e.g., Laemmli'sbuffer) containing both detergent (e.g., sodium dodecyl sulfate) and areducing agent (e.g., DTT or beta-mercaptoethanol). The sample can thenbe subjected to SDS-polyacrylamide gel electrophoresis (PAGE). PAGEresolved proteins, separated by size, can then be transferred to afilter membrane (e.g., nitrocellulose) and subjected to western blottechniques using antibodies specific to neublastin. The level ofneublastin in a sample can be determined by comparison to a control orreference sample containing a known amount of neublastin. In analternative embodiment of the western technique, sometimes called adot-blot method, the protein sample can be directly adhered to a filtermembrane without prior SDS-PAGE resolution.

Immunoassays using the anti-neublastin antibodies described herein canbe performed completely in solution. Examples of solution-based assaysimmunoassays include fluorescence resonance energy transfer (FRET)-basedimmunoassays, which use two detection moieties and entail theradiationless transfer of energy from a donor molecule to an acceptormolecule. The donor molecule can be a dye or chromophore that initiallyabsorbs energy and the acceptor can be a chromophore to which the energyis subsequently transferred (called a donor/acceptor pair). Thisresonance interaction occurs over greater than inter-atomic distances,without conversion to thermal energy and without any molecularcollision. Due to its sensitivity to distance, FRET is extremely usefulin investigating protein-protein interactions and enzymatic reactions. Acompetition-based FRET immunoassay can be used to detect a neublastinpolypeptide in a sample, wherein a specific anti-neublastin antibody iscoupled to a first detection moiety (e.g., donor or acceptor molecule).The neublastin polypeptide containing sample can then be mixed with aneublastin polypeptide reference (i.e., standard) conjugated to a seconddetection moiety (e.g., acceptor or donor molecule where appropriate),which is also recognized by the anti-neublastin antibody and competeswith the neublastin in a sample for binding to the anti-neublastinantibody. Both the sample neublastin polypeptide and theneublastin-detection standard are incubated with the antibody for a timesufficient for the antibody to bind to neublastin or the standard.Binding of the neublastin standard to the antibody, and the detectionsignal produced by the assay, is dependent on the level of neublastinpresent in the sample (i.e., the higher the amount of neublastinpolypeptide in a sample, the lower the signal produced in the assay).Quantitation of the amount of neublastin in a sample can be determinedby comparing the amount of signal produced by the neublastin-detectionmoiety standard in the absence of sample neublastin polypeptide relativeto the signal produced from samples in which sample neublastin is mixedwith the standard.

For purposes of detection, the donor molecule of the FRET immunoassaycan be a fluorescent agent such a europium, terbium, green-fluorescentprotein, or a fluorescent dye, and the acceptor molecule can be, forexample, allophycocyanin (APC). These donor and acceptor molecules canbe directly attached to the antibody or neublastin standards, or can beconjugated to a first member of a specific binding pair (e.g., biotin orstreptavidin) and the detection moiety thus coupled to a second memberof a binding pair (e.g., streptavidin or biotin).

Methods of assessing the level of a neublastin polypeptide in a samplecan be quantitative, semi-quantitative, or qualitative. Thus, forexample, the level of neublastin in a sample can be determined as adiscrete value. For example, where quantitative immunoassays arenecessary, the level of neublastin can be measured as a numerical valueby correlating the detection signal derived from the quantitative assayto the detection signal of a known concentration of neublastinpolypeptide or the signal presence of neublastin in a reference sampleprovided from a second subject. Alternatively, the level of neublastinpolypeptide can be assessed using any of a variety ofsemi-quantitative/qualitative systems. Thus, the level of expression ofneublastin polypeptide useful in the immunoassay in a sample can beexpressed as, for example, (a) one or more of “excellent”, “good”,“satisfactory”, “unsatisfactory”, and/or “poor”; (b) one or more of“very high”, “high”, “average”, “low”, and/or “very low”; or (c) one ormore of “++++”, “+++”, “++”, “+”, “+/−”, and/or “−”. In this aspect,where it is also desired, the level of neublastin polypeptide can beexpressed relative to the neublastin levels of neublastin polypeptide ina reference sample from a second subject.

A neublastin-containing sample can be of unknown concentration ofneublastin or a known concentration of neublastin (e.g., a neublastinstandard for the assay). Assay standards, such as those useful in aneublastin immunoassay, are commonly purified proteins (e.g., arecombinant neublastin or neublastin purified from a natural source) atknown concentrations, solubilized and/or diluted into an appropriatebuffer used in the immunoassay (e.g., PBS, TBST, Water, HEPES). Theneublastin-containing sample can be a biological fluid such as serum,blood, urine, semen, cerebral-spinal fluid, saliva, or lacrimalsecretions. The sample can be obtained from a variety of sources, e.g.,cell culture or a mammal (such as a mouse or a human). Theneublastin-containing sample can contain exogenous and/or endogenousforms of neublastin.

Pharmaceutical Compositions

The anti-neublastin antibodies and antibody fragments described hereincan be administered to a mammalian subject (e.g., a human) alone or in amixture. For example, the antibodies and antibody fragments can beadministered in the presence of a pharmaceutically acceptable excipientor carrier, such as physiological saline. The excipient or carrier canbe selected on the basis of the mode and route of administration.Suitable pharmaceutical carriers are described in Remington'sPharmaceutical Sciences (E. W. Martin) and in the USP/NF (United StatesPharmacopeia and the National Formulary).

A pharmaceutical composition is generally formulated to be compatiblewith its intended route of administration. Examples of routes ofadministration of an anti-neublastin antibody or antibody fragmentinclude, e.g., intravenous, intradermal, subcutaneous, oral (e.g.,inhalation), transdermal (topical), transmucosal, and rectaladministration. Solutions or suspensions used for parenteral,intradermal, or subcutaneous application can include the followingcomponents: a sterile diluent such as water for injection, salinesolution, fixed oils, polyethylene glycols, glycerine, polypropyleneglycol or other synthetic solvents; antibacterial agents such as benzylalcohol or methyl parabens; antioxidants such as ascorbic acid or sodiumbisulfite; chelating agents such as ethylenediaminetetraacetic acid;buffers such as acetates, citrates or phosphates and agents foradjustment of tonicity such as sodium chloride or dextrose. pH can beadjusted with acids or bases, such as hydrochloric acid or sodiumhydroxide. The parenteral preparation can be enclosed in ampoules,disposable syringes, or multiple dose vials made of glass or plastic.

A pharmaceutical composition may include a “therapeutically effectiveamount” or a “prophylactically effective amount” of an anti-neublastinantibody or antibody fragment described herein. As used herein,“therapeutically effective amount” means an amount effective, atdosages, and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody fragment can vary according to factors such as the diseasestate, age, sex, and weight of the individual, and the ability of theantibody, antibody derivative, or antigen-binding polypeptide to elicita desired response in an individual. When a therapeutically effectiveamount is administered, any toxic or detrimental effects of the antibodyor antibody fragment are outweighed by the therapeutically beneficialeffects. As used herein, “prophylactically effective amount” means anamount effective, at dosages, and for periods of time necessary, toachieve the desired prophylactic result.

Dosage regimens can be adjusted to provide the optimum desired response,e.g., a therapeutic or prophylactic response. For example, in someembodiments a single bolus is administered. In other embodiments,several divided doses are administered over time. The dose can bereduced or increased proportionately, as indicated by the exigencies ofthe situation. It is advantageous to formulate parenteral compositionsin dosage unit form for ease of administration and uniformity of dosage.As used herein, “dosage unit form” means physically discrete unitssuitable as unitary dosages for the mammalian subjects to be treated,with each containing a predetermined quantity of active ingredientcalculated to produce the desired therapeutic effect in association withthe required pharmaceutical carrier.

Exemplary, non-limiting ranges for a therapeutically or prophylacticallyeffective amount of an antibody or antibody fragment are 0.1-100 mg/kg,0.5-50 mg/kg, more 1-20 mg/kg, and 1-10 mg/kg. Dosage values may varywith the type and severity of the condition being treated. For anyparticular subject, specific dosage regimens can be adjusted over timeaccording to the individual need and the professional judgment of theperson administering or supervising the administration of thecompositions. It is to be understood that dosage ranges set forth hereinare exemplary only and are not intended to limit the scope of theclaimed invention.

Parenteral injectable administration can be used for subcutaneous,intramuscular, or intravenous injections and infusions. Additionally,one approach for parenteral administration employs the implantation of aslow-release or sustained-released systems, which assures that aconstant level of dosage is maintained, according to U.S. Pat. No.3,710,795, incorporated herein by reference.

Subjects intended for treatment or prophylaxis include but are notlimited to humans, nonhuman primates, sheep, horses, cattle, goats,pigs, dogs, cats, rabbits, guinea pigs, hamsters, gerbils, rats, andmice.

Methods of Antagonizing Neublastin Activity

As detailed in the Examples, the P1H1.G7 and P3B3.6 monoclonalantibodies bind to neublastin and the binding of anti-neublastinmonoclonal antibodies to neublastin was shown to inhibit its ability totrigger the neublastin-mediated signaling cascade and induce RETautophosphorylation. As a result, anti-neublastin antibodies describedherein can be used as antagonists of neublastin bioactivity. Inparticular, the anti-neublastin antibodies can be administered to amammal (e.g., a human) in an amount effective to reduce or eliminate theability of endogenous, wild-type neublastin to bind to a complexcontaining RET and GFRalpha3 and induce dimerization andautophosphorylation of RET. Antagonism of neublastin bioactivity can beparticularly useful in the treatment of cancers, such as pancreaticcancers where neublastin promotes cancer cell invasion. Additionalexemplary cancers that can be treated with an anti-neublastin antibodyor antigen-binding fragment thereof described herein include cancers ofthe gastrointestinal tract (e.g., esophageal or colon cancer) as well ascancers of the bladder, breast, connective tissue, kidney, lung (e.g.,small cell lung carcinoma), lymph node, ovary, skin, stomach, testis,and uterus.

The following are examples of the practice of the invention. They arenot to be construed as limiting the scope of the invention in any way.

EXAMPLES Example 1 Development of Anti-Neublastin Monoclonal Antibodies

Eight week old female RBF mice (Jackson Labs, Bar Harbor, Me.) wereimmunized intraperitoneally with an emulsion containing either (i) asoluble Chinese Hamster Ovary (CHO) cell-expressed human neublastin(hNBN104)-hIgG1 Fc recombinant protein or (ii) an E. coli expressedhNBN113 molecule chemically conjugated to keyhole limpet hemocyanin(KLH). Both preparations were emulsified into Freund's complete adjuvant(FCA, Sigma Chemical Co., St. Louis, Mo.) for primary immunization andsubsequently emulsified into Freund's incomplete adjuvant (IFA) forremaining booster immunizations. Further details on the protein hNBNpreparations, immunizations, and hybridoma preparations are providedbelow.

hNBN-hIgG1 Fc protein or E. coli hNBN-KLH was diluted into phosphatebuffered saline (PBS), pH 7.2 at a concentration of approximately 2mg/ml. An equal volume of FCA was added to the protein prior toemulsification and immunization. For the primary immunization, eachmouse was intraperitoneally administered 50 μL containing 50 μg ofemulsified hNBN antigen. All subsequent immunizations were similarlydosed using either IFA or RIBI adjuvant (Sigma Chemical Co., St. Louis,Mo.). Booster immunizations were administered every two to three weeks.

Serum samples from immunized mice were collected before the firstimmunization, 7 days post the booster immunization, and again prior tolymphocyte cell fusions via the retro orbital plexus collection method.Serum titers were measured using an ELISA assay described below.

A mouse was selected for hybridoma development from an animal cohortimmunized with the CHO expressed hNBN-IgFc. Several antibody-producingB-cell clones were derived from splenocytes isolated from this mouse.The P1H1.G7 clone produced an antibody able to bind both rat and humanneublastin proteins, as determined by ELISA.

Isolated splenocytes from a mouse immunized with the E. coli-expressedhNBN-KLH were used to generate B-cell hybridoma lines expressinganti-neublastin antibodies. The monoclonal antibody P3B3.6 clone wasselected on the basis of its ability to specifically bind to E. coli ratNeublastin protein. The P3B3.6 monoclonal antibody was also found tobind to human neublastin.

The development of the P1H1.G7 and P3B3.6 hybridomas is described withadditional detail in the following sections.

Keyhole Limpet Hemocyanin Linkage Reaction

One-milliliter E. coli expressed hNBN (1.7 mg/ml in L-arg buffer) wasdialyzed into MES buffer, pH 4.7 containing 0.9% NaCl. Two milligramsKLH (CalbioChem, Inc.) in similar MES buffer was mixed with the hNBN andstirred for 1 hour into a homogenous mixture. Four milligrams ofethylene dichloride (EDC, Pierce Chemical) was added to the proteinmixture and allowed to mix for 2 hours at ambient temperature. Thereaction was stopped by adding 100 μl saturated L-glycine (SigmaChemical) solution and allowed to stir for 1 hour. The conjugatedmixture was passed over a 30×10 ml 10 DG Econopack column (BioRad) thatwas equilibrated with 5 column volumes PBS. Fractions containing KLHconjugate were identified and pooled and prepared for immunizations.

Cell Lines and Media

FL653 (an APRT⁻ derivative of a Ig⁻/HGPRT⁻ Balb/c mouse myeloma cellline) and SP2/0-Ag14 (an Ig⁻/HGPRT⁻ Balb/c mouse myeloma cell line) wereeach cultured in 10% fetal bovine serum in Dulbecco's modified Eagle'smedium (DMEM, Sigma Chemical Co., St. Louis, Mo.) containing 4500 mg/Lglucose, L-glutamine, and 20 μg/ml 8-azaguanine (Sigma Chemical Co.) forat least 10 days prior to lymphocyte cell fusion experiments. Myelomacells were cultured in a Series II™ model water jacketed incubator(Forma Scientific, Marietta, Ohio) which had been programmed to maintaina 37° C., 98% humid environment with a 7% CO₂ in air atmosphere.

Solid Phase Assay (ELISA)

Maxisorp, 96-well microtiter plates (Nunc) were coated overnight with 50ul/well of a 2 ug/ml sample of either the unconjugated E. coli expressedhNBN or the CHO expressed hNBN-hIgG1 Fc recombinant protein in 100 mMSodium Phosphate Buffer, pH 8.0. Plates were emptied and washed threecycles with a solution of 0.05% Tween-20 in deionized water using anEmbla automated plate washer (Skatron). Following the wash procedure,plates were filled with a 0.5% Bovine Serum Albumin (BSA) in PBSblocking solution and allowed to incubate 1 hour 37° C. Following theplate block, plates were flicked clear and 50 μl serial dilutions ofserum and preserum samples from immune mice (or from monoclonalproducing hybridoma supernatants) were applied to the ELISA plate andallowed to incubate 1 hour at 37° C. (diluted 1:5). Plates were washedas described above and then applied with 50 μl diluted goat anti-mouseIgG-HRP (Jackson Labs) and again allowed to incubate 1 hour at 37° C.Plates were washed followed by adding 50 μl Ultra TMB (Pierce) substrateand allowed to catalyze for approximately 10 minutes. The enzymereaction was killed by adding equal volume 2.0 N H2SO4 and plates wereread at 450 nm on a Spectramax 384 Plus (Molecular Devices) automatedplate scanner. ELISA analysis was completed using Softmax Pro (MolecularDevices) absorbance analysis software.

Lymphocyte Cell Fusion

Mice that tested positive for expression of antibodies specific toeither of the two neublastin antigen forms (CHO cell-expressed hNBN104and E. coli-expressed hNBN113 polypeptide form) were sacrificed, andtheir splenic B-lymphocytes were aseptically harvested. SplenicB-lymphocytes were washed and prepared for use in PEG mediatedlymphocyte somatic cell fusions, being fused to either the FL653 orSP2/0-Ag14 myeloma (Kennet, et al. 1982. Plenum Press, NY). Fused cellswere plated into 24-well sterile tissue culture plates (Corning GlassWorks, Corning, N.Y.) and fed with Adenine, Aminopterin and Thymidine(AAT) or Hypoxanthine, Aminopterin and Thymidine-containing culturemedia, for FL653 or SP2/0-Ag14 myeloma based fusions respectively. Thecell culture environment was maintained at 37° C., 98% humidity and a7.2% CO₂ in air atmosphere.

After 10 days, AAT or HAT resistant cultures were isolated and screenedby ELISA for immunoreactivity specific to both eukaryotic andprokaryotic expression forms of the neublastin protein. Positivecultures were subsequently cloned, expanded and frozen. Cloning wasperformed by limiting dilution (approximately 1 cell/well) andmicroscopically scored upon growth to assure integrity of the selectedclones. Clones that screened positive on both ELISA format assays (i.e.,positive for unconjugated E. coli expressed hNBN or the CHO expressedhNBN-hIgG1 Fc recombinant protein) were expanded for freezing, subclasscharacterized using IsoStrip (Roche), and assayed for monoclonalproduction level.

Example 2 Sensitive Method for Detecting Exogenous Neublastin

In an assay termed the “P3B3 assay,” a P3B3.6 antibody was used as acapturing antibody and a biotinylated P3B3.6 antibody was used fordetection. This assay, which is sensitive to approximately 0.05-0.1ng/ml and detects exogenous, recombinant neublastin but not endogenousneublastin, is described in detail below.

P3B3.6 antibodies were adhered to 96-well polystyrene assay plates(COSTAR, Corning Inc.) by incubating 50 ng/well of antibody (captureantibody) in PBS for 40 minutes at 37° C. All incubation steps werecarried out with constant shaking, unless otherwise indicated. Followingantibody binding, the plates were blocked with 1× casein-TBST solution(Vector Laboratories, Inc.; 0.08% casein by weight plus TBST: 25 mMTris-HCl, pH 8.0, NaCl 125 mM, 0.1% Tween-20 by weight) and incubatedfor 1 hour at room temperature. The plates were washed three times withTBST at room temperature.

Recombinant neublastin (hNBN104 expressed in CHO cells) standards wereprepared in normal rat serum or TBST with 0.05% bovine serum albumin(BSA) at the following concentrations: 4 ng/mL, 2 ng/mL, 1 ng/mL, 500pg/mL, 250 pg/mL, 125 pg/mL, 62.5 pg/mL, and 31.25 pg/mL. Into eachP3B3.6 antibody-coated well (each reaction), 90 μL of TBST was addedalong with 10 μL of one of the above neublastin standards, or test sera.The plates were incubated overnight at 4° C. Following the incubation,wells were washed three times with TBST at room temperature.

The detection antibody (biotinylated P3B3.6) was diluted to 0.2 μg/mL in1× casein-TBST, and 100 μL of this diluate was added to each wellfollowed by a 1 hour incubation at room temperature. The plates werethen washed three times with TBST. Antibody-captured neublastin wasdetected using a streptavidin-conjugated horseradish peroxidase (HRP)enzyme reagent. Streptavidin-HRP was diluted 1,000 fold in 1×casein-TBST, and 100 μL was added to each well, followed by a 30 minuteincubation at room temperature. The plates were washed three times withTB ST. HRP enzyme substrate (Luminol/Enhancer solution and StablePeroxide solution, Pierce Labs) was added at 100 μL per well andincubated in the dark for 1 minute. Resulting light emissions weremeasured using a luminometer (Tropix TR717, PE Applied Biosystems).

Each of the three neublastin standard sample replicates was averaged andtheir respective standard deviation and % CV values calculated. Thevalues from the averaged standard samples were fitted with a 4-parametersigmoidal logistic curve (FIG. 2). When the linear portion of the P3B3assay was expanded, the assay appeared to be linear down to 0.1 ng/mlwith a potential to go as low as 0.03 ng/ml (FIG. 3).

To further explore the ability of the P3B3 assay to reproduciblyidentify the correct serum concentration of neublastin, an experimentwas conducted to test “sham” unknowns against the neublastin standards.The concentrations of the sham unknowns were estimated from the standardcurve and compared to the known amounts in each well. To determine thepercentage recovery for each sham unknown, the experimentally determinedvalues were divided by the expected values. Overall recovery averaged104% with a standard error of +/−13%. Greatest data agreement occurredin the middle range neublastin concentrations, with outlying valuespredominantly occurring at neublastin concentrations below 0.125 ng/mL.Concentrations as low as 0.1 ng/mL were still within the linear range.

Example 3 Sensitive Method for Monitoring Levels of Exogenous Neublastinin an Animal Following Administration

The P3B3 assay (described in Example 2) was evaluated for its ability todetect neublastin in serum. Rats were subcutaneously injected with 1mg/kg of CHO cell-derived, recombinant human neublastin (hNBN104). Atvarious time points post-injection, serum samples were collected andassayed for neublastin protein concentration. The assay clearlyidentified exogenous human neublastin that demonstrated an expectedkinetic response (FIG. 4), indicating that the P3B3 assay is useful indetecting low levels of administered, recombinant human neublastin.

Example 4 Sensitive Method for Detecting Exogenous and EndogenousNeublastin

In an assay termed the “P1H1 assay,” a P3B3.6 monoclonal antibody wasused as a capturing antibody and a biotinylated P1H1.G7 monoclonalantibody was used for detection. Several assay parameters were evaluatedusing different combinations of these two monoclonals, including biotinratios, pH, detergent, incubation time, and sample volume.

P1H1.G7 was biotinylated at three different ratios, 1:5, 1:10, and 1:20.Based on the linearity and signal to noise ratio, the 1:10 antibody tobiotin ratio was chosen. Tween-20 added to the incubation bufferincreased the assay's background signal in this assay. However, PBSbuffer gave lower background than Hepes buffer, while pH did not seem tohave a significant effect. Serum samples were incubated in P3B3.6monoclonal antibody-coated wells for different times, ranging from 1.0hour to overnight (about 18 hours). Overnight incubation gave bettersensitivity. Different serum sample volumes ranging from 5 ul to 100 ulwere tested in the assay and it was determined that 10 ul serum providedthe best sensitivity of 1 ng/ml with a minimal matrix effect.

The P1H1 assay provided a sensitivity of approximately 1 ng/ml.Furthermore, the P1H1 assay was found to detect both recombinant,exogenous neublastin as well as endogenous neublastin. This contrastswith the P3B3 assay (Examples 2 and 3), which detected only exogenousneublastin.

Example 5 The Anti-hNBN P3B3.6 Antibody can Block NBN-Mediated RETActivation

The anti-NBN monoclonal antibodies were evaluated for their ability toantagonize NBN-mediated RET activation in murine cells. NB41A3-mRL3adherent murine neuroblastoma cells, which express c-RET and GFRalpha3polypeptides, were seeded at 2×10⁵ cells/well in 24-well tissue cultureplates and grown in DMEM to a confluency of 75%. Next, 75 nM samples ofrat NBN were preincubated at room temperature for one hour in eitherDMEM alone, or in DMEM containing various concentrations (approximately0.01 to 1,000 nM) of each of P1H1.G7 monoclonal antibody, P3B3.6monoclonal antibody, or a control antibody (7P1H10.3). Following thepreincubation, each of above rat NBN samples were added to the medium ofNB41A3-mRL3 cell cultures, and inbuted with the cells for 10 minutes at37° C. The cells were washed, lysed, and c-RET polypeptide contained inthe cell lysates was captured on an ELISA plate coated with anti-RETmonoclonal antibodies. Phosphorylated forms of c-RET captured on theplate were detected using a horseradish peroxidase-coupledanti-phosphotyrosine monoclonal antibody (AA.GE7.3). Preincubation ofrat NBN with the P3B3.6 monoclonal antibody inhibited the ability of NBNto induce phosphorylation of c-RET in murine cells (FIG. 5). Theseresults indicated that the P3B3.6 monoclonal antibody antagonizesNBN-mediated activation of c-RET.

Other Embodiments

While the invention has been described in conjunction with the detaileddescription thereof, the foregoing description is intended to illustrateand not limit the scope of the invention, which is defined by the scopeof the appended claims. Other aspects, advantages, and modifications arewithin the scope of the following claims.

1. An isolated antibody or antigen-binding fragment thereof thatselectively binds to the polypeptide of SEQ ID NO:1 on the same epitopeas the antibody produced by the hybridoma deposited in the ATCC underAccession No. PTA-7624 or PTA-7625.
 2. An isolated antibody orantigen-binding fragment thereof that selectively binds to thepolypeptide of SEQ ID NO:1 and crossblocks binding of the antibodyproduced by the hybridoma deposited in the ATCC under Accession No.PTA-7624 or PTA-7625.
 3. The antibody or antigen-binding fragmentthereof of claim 1, wherein the antibody or antigen-binding fragmentthereof is a monoclonal antibody, a polyclonal antibody, a humanizedantibody, a fully human antibody, a single chain antibody, a chimericantibody, an Fab fragment, an F(ab′)2 fragment, an Fab′ fragment, an Fscfragment, or an Fv fragment.
 4. An antibody produced by the hybridomadeposited in the ATCC under Accession No. PTA-7624 or PTA-7625.
 5. Aconjugate comprising the antibody or antigen-binding fragment thereof ofclaim 1 linked to a detectable label.
 6. The conjugate of claim 5,wherein the detectable label is horseradish peroxidase or alkalinephosphatase.
 7. The conjugate of claim 5, wherein the detectable labelis a fluorescent or radio-labeled marker.
 8. A conjugate comprising theantibody or antigen-binding fragment thereof of claim 1 linked to asolid-phase matrix.
 9. The conjugate of claim 8, wherein the solid-phasematrix is a multi-well assay plate, sepharose, agarose, or a magneticbead.
 10. A pharmaceutical composition comprising the antibody orantigen-binding fragment thereof of claim 1 and a pharmaceuticallyacceptable carrier.
 11. An isolated cell that produces the antibody orantigen-binding fragment thereof of claim
 1. 12. The hybridoma depositedin the ATCC under Accession No. PTA-7624 or PTA
 7625. 13. A method ofdetermining the presence or amount of a neublastin polypeptide in asample, the method comprising: contacting a sample with the antibody orantigen-binding fragment thereof of claim 1 for a time sufficient forthe antibody or antigen-binding fragment thereof to bind to a neublastinpolypeptide, if present in the sample; and detecting the presence oramount of the antibody or antigen-binding fragment thereof bound to theneublastin polypeptide to thereby determine the presence or amount ofthe neublastin polypeptide in the sample.
 14. The method of claim 13,wherein the method comprises: prior to the detecting step, contactingthe sample with a second antibody or antigen-binding fragment thereofthat selectively binds to the polypeptide of SEQ ID NO:1 for a timesufficient for the second antibody or antigen-binding fragment thereofto bind to the neublastin polypeptide bound to the antibody orantigen-binding fragment thereof of any of claims 1 to 4; and detectingthe presence or amount of the second antibody or antigen-bindingfragment thereof bound to the neublastin polypeptide.
 15. A method ofdetermining the presence or amount of a neublastin polypeptide in asample, the method comprising: contacting a sample with a first antibodyor antigen-binding fragment thereof that selectively binds to thepolypeptide of SEQ ID NO:1 on the same epitope as the antibody producedby the hybridoma deposited in the ATCC under Accession No. PTA-7625 fora time sufficient for the first antibody or antigen-binding fragmentthereof to bind to a neublastin polypeptide, if present in the sample;contacting the sample with a second antibody or antigen-binding fragmentthereof that selectively binds to the polypeptide of SEQ ID NO:1 on thesame epitope as the antibody produced by the hybridoma deposited in theATCC under Accession No. PTA 7624 for a time sufficient for the secondantibody or antigen-binding fragment thereof to bind the neublastinpolypeptide, if bound to the first antibody or antigen-binding fragmentthereof; and detecting the presence or amount of the second antibody orantigen-binding fragment thereof bound to the neublastin polypeptide, tothereby determine the presence or amount of the neublastin polypeptidein the sample.
 16. The method of claim 15, wherein the first antibody isproduced by the hybridoma deposited in the ATCC under Accession No.PTA-7625.
 17. The method of claim 15, wherein the second antibody isproduced by the hybridoma deposited in the ATCC under Accession No.PTA-7624.
 18. A method of determining the presence or amount of aneublastin polypeptide in a sample, the method comprising: contacting asample with a first antibody or antigen-binding fragment thereof thatselectively binds to the polypeptide of SEQ ID NO:1 on the same epitopeas the antibody produced by the hybridoma deposited in the ATCC underAccession No. PTA-7625 for a time sufficient for the first antibody orantigen-binding fragment thereof to bind to a neublastin polypeptide, ifpresent in the sample; contacting the sample with a second antibody orantigen-binding fragment thereof that selectively binds to thepolypeptide of SEQ ID NO:1 on the same epitope as the antibody producedby the hybridoma deposited in the ATCC under Accession No. PTA 7625 fora time sufficient for the second antibody or antigen-binding fragmentthereof to bind to the neublastin polypeptide, if bound to the firstantibody or antigen-binding fragment thereof; and detecting the presenceor amount of the second antibody or antigen-binding fragment thereofbound to the neublastin polypeptide, to thereby determine the presenceor amount of the neublastin polypeptide in the sample.
 19. The method ofclaim 18, wherein the first antibody is produced by the hybridomadeposited in the ATCC under Accession No. PTA-7625.
 20. The method ofclaim 18, wherein the second antibody is produced by the hybridomadeposited in the ATCC under Accession No. PTA-7625.
 21. The method ofclaim 13, wherein the method is a sandwich-based immunoassay.
 22. Themethod of claim 13, wherein the sample is obtained from a mammal. 23.The method of claim 22, wherein the neublastin polypeptide isendogenous, wild-type neublastin.
 24. The method of claim 13, whereinthe neublastin polypeptide comprises an amino acid sequence that is atleast 80% identical to amino acids 15-113 of SEQ ID NO:1 and thepolypeptide, when dimerized, binds to a complex containing GFRalpha3 andRET, and wherein the sample is obtained from a mammal to which theneublastin polypeptide has been administered.
 25. The method of claim24, wherein the amino acid sequence is at least 90% identical to aminoacids 15-113 of SEQ ID NO:1.
 26. The method of claim 24, wherein theamino acid sequence is at least 95% identical to amino acids 15-113 ofSEQ ID NO:1.
 27. The method of claim 24, wherein the amino acid sequenceis at least 98% identical to amino acids 15-113 of SEQ ID NO:1.
 28. Themethod of claim 24, wherein the polypeptide comprises amino acids 15-113of SEQ ID NO:1, amino acids 15-113 of SEQ ID NO:2, amino acids 15-113 ofSEQ ID NO:3, amino acids 15-113 of SEQ ID NO:4, amino acids 15-113 ofSEQ ID NO:5, amino acids 15-113 of SEQ ID NO:8, or amino acids 15-113 ofSEQ ID NO:9.
 29. The method of claim 24, wherein the polypeptidecomprises the amino acid sequence of SEQ ID NO:1, the amino acidsequence of SEQ ID NO:2, the amino acid sequence of SEQ ID NO:3, theamino acid sequence of SEQ ID NO:4, the amino acid sequence of SEQ IDNO:5, the amino acid sequence of SEQ ID NO:8, or the amino acid sequenceof SEQ ID NO:9.
 30. The method of claim 24, wherein the polypeptidecomprises amino acids 10-113 of SEQ ID NO:1.
 31. The method of claim 13,wherein the sample is blood, serum, saliva, semen, urine, lacrimalfluid, or cerebral spinal fluid.
 32. A method of antagonizing neublastinactivity, the method comprising administering to a mammal an amount ofthe antibody or antigen-binding fragment thereof of claim 1 effective toreduce or eliminate the ability of endogenous, wild-type neublastin tobind to a complex comprising RET and GFRalpha3 and induce dimerizationand autophosphorylation of RET.
 33. A method of treating a cancer, themethod comprising administering to a mammal having a cancer apharmaceutical composition comprising a therapeutically effective amountof the antibody or antigen-binding fragment thereof of claim
 1. 34. Themethod of claim 33, wherein the cancer is a pancreatic cancer.
 35. Themethod of claim 22, wherein the mammal is a human.